Clinical applications of stem cell-derived exosomes

Although stem cell-based therapy has demonstrated considerable potential to manage certain diseases more successfully than conventional surgery, it nevertheless comes with inescapable drawbacks that might limit its clinical translation. Compared to stem cells, stem cell-derived exosomes possess numerous advantages, such as non-immunogenicity, non-infusion toxicity, easy access, effortless preservation, and freedom from tumorigenic potential and ethical issues. Exosomes can inherit similar therapeutic effects from their parental cells such as embryonic stem cells and adult stem cells through vertical delivery of their pluripotency or multipotency. After a thorough search and meticulous dissection of relevant literature from the last five years, we present this comprehensive, up-to-date, specialty-specific and disease-oriented review to highlight the surgical application and potential of stem cell-derived exosomes. Exosomes derived from stem cells (e.g., embryonic, induced pluripotent, hematopoietic, mesenchymal, neural, and endothelial stem cells) are capable of treating numerous diseases encountered in orthopedic surgery, neurosurgery, plastic surgery, general surgery, cardiothoracic surgery, urology, head and neck surgery, ophthalmology, and obstetrics and gynecology. The diverse therapeutic effects of stem cells-derived exosomes are a hierarchical translation through tissue-specific responses, and cell-specific molecular signaling pathways. In this review, we highlight stem cell-derived exosomes as a viable and potent alternative to stem cell-based therapy in managing various surgical conditions. We recommend that future research combines wisdoms from surgeons, nanomedicine practitioners, and stem cell researchers in this relevant and intriguing research area.


INTRODUCTION
Stem cells are a population of undifferentiated cells with unique abilities to self-renew and recreate functional tissues.They are primarily classified by their differentiation potential, origin and lineage progression.According to their potency, stem cells can be totipotent, pluripotent, multipotent, oligopotent and unipotent. 1tem cells exist both in embryos and adult cells.Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are best examples of pluripotent stem cells, 2 whereas adult multipotent stem cells are exemplified by hematopoietic stem cells (HSCs), 3 mesenchymal stem cells (MSCs), 4 neural stem cells (NSCs), 5 and endothelial stem/progenitor cells (EPCs) 6 (Fig. 1a).All these subtypes of stem cells have been extensively trialed for the treatment of human diseases.
Stem cell-based therapy, as a modality of regenerative medicine, has generated tremendous attention, as it offers new options for patients suffering from previously incurable diseases.Subsequently, thousands of related clinical trials have been registered, covering a wide spectrum of medical problems, such as musculoskeletal and neurological disorders, immune diseases, hematological dysfunctions, and degenerative conditions. 7However, some trials have failed to show any benefit in the clinic.This is likely due to the inevitable limitations of stem cell therapy, such as infusion toxicity, immunogenicity, tumorigenic potentials and ethical issues. 8xosome, secreted by almost all cell types including stem cells (Fig. 1a), has been posited as a safer and more versatile alternative to stem cell therapy. 9xosomes are nanoscale, spherical, and lipid bi-layered single membrane extracellular vesicles, which act as intercellular messengers. 10Exosomes have been regarded as miniature versions of their parental cells, partially because exosomes from a certain cell type provide cell-specific or unique sets of biomolecules.In addition, the stem cells have been found to function in a paracrine fashion through their soluble secretome including exosomes. 11In other words, stem cell-derived exosomes (SC-Exo) inherit similar therapeutic effects from their parental cell of origin, e.g., anti-inflammation, immunomodulation and tissue regeneration. 12Collectively, stem cell-derived exosomes are a potent surrogate for stem cell therapy without exhibiting the disadvantages their cellular counterparts present 13 (Table 1).
Prior to clinical applications, exosomes must be prepared and optimized in terms of production, purification, and modification (Sections 2.3 and 2.4).A wide range of medical reviews analyzing these upstream measures of exosome therapy have been published in recent years.Nevertheless, some research avenues remain under-investigated: in particular, systematic investigation dedicated to downstream clinical applications is lacking, especially from a surgical perspective.Tissues that have been damaged, whether by disease or a surgeon's scalpel, respond by inflammatory and regenerative dynamics, 14 making surgery a perfect arena for stem cell-derived exosome therapy. 15][18] In this work, we will dissect relevant publications from the last five years in order to present a comprehensive, up-to-date, specialty-specific and disease-oriented review (Fig. 2).Our aim is to bridge the gap that currently exists between surgeons, nanomedicine practitioners, and stem cell researchers.

GENERAL BACKGROUND OF EXOSOMES AND EXOSOME THERAPY
Biogenesis, composition, and uptake of exosomes Exosomes differ from other types of primary extracellular vesicles (e.g., apoptotic bodies and microvesicles) in terms of size, content, and production mechanism. 19The most popularly accepted mechanism of exosome formation, i.e., an endosomal route, is as follows (Fig. 1a).The initial endosomes are produced by cell membrane invagination during which the bioactive substances begin to accumulate within the early sorting endosomes.The late sorting endosomes then form multivesicular bodies (MVBs) after a second indentation.Finally, the MVBs fuse with the cell membrane, releasing the carried exosomes to the outside.Nonendosomal route of exosome biogenesis, such as plasma membrane budding, has also been reported. 20s the three major exosome databases (i.e., ExoCarta, Vesiclepedia, and EVpedia) summarize, exosomes contain numerous molecules, including proteins, glycoconjugates, lipids, nucleic acids, metabolites, and other bioactive substances (Fig. 1b).The examples of each category and the corresponding functions have been thoroughly reviewed elsewhere. 21,22On the one hand, exosomes comprise a complex protein network including external proteins (e.g., tetraspanins, antigen-presenting complexes, and adhesion molecules) and internal proteins (e.g., heat shock proteins, ESCRT machinery, cytokines and chemokines, and membrane transporters). 23On the other hand, as the most abundant in human exosomal nucleic acids, microRNA (mRNA) could participate in hematopoiesis, exocytosis, and nerve and vascular regeneration through exosome-mediated cellular communication. 24here are various uptake mechanisms once exosomes reach the recipient cell, all of which can be categorized into membrane fusion, receptor interaction, and internalization 21 (Fig. 1b).Finally, the exosomal cargos are released into the cytoplasm, the process of which depends on the source of the exosome, nature of the cargo, and the metabolic state of the recipient cell. 25The entire lifecycle from exosome biogenesis to uptake and intracellular signaling can be tracked using fluorescent, luminescent, and radioactive techniques. 26,27urce and classification of exosomes Depending on whether exosomes have been artificially modified, they are broadly classified into natural exosomes and engineered exosomes (Section 2.4).Depending on the species of origin, exosomes are divided into animal-derived and plant-derived exosomes.Currently, exosomes are mainly classified according to the type of their parental cells.Almost all types of human cells can produce exosomes.These include, but are not limited to, macrophages, dendritic cells (DCs), platelets, stem cells, and even tumor cells 28 (Fig. 1a).
For example, macrophage-derived exosomes contribute to disease progression (e.g., diabetes, atherosclerosis and heart failure) 29 and disease treatment (e.g., cutaneous wound, inflammatory bowel disease, and fungal and viral infection). 30However, they seem to play paradoxical roles in suppressing and promoting tumors. 31Like DCs, DC-derived exosomes (Dex) could also interact with immune cells (e.g., T cells, B cells, and NK cells) through their surface proteins such as major histocompatibility complexes (MHCs). 32Some preclinical and clinical trials have demonstrated the effectiveness and safety of Dex-based immunotherapy for cancers. 33Furthermore, tumor-derived exosomes (Tex) not only are involved during tumor proliferation, invasion, metastasis, and immunity but also can be used as biomarkers for cancer diagnosis and treatment. 34Lately, Tex has been used as an anti-tumor drug and an antigen presenter for DC vaccination, serving as a promising cell-free cancer immunotherapy. 35Finally, the clinical applications of stem cell-derived exosomes will be discussed in detail in the following sections.
Exosomes can be found in all body fluids such as blood, saliva, urine, plasma, tears, semen, amniotic fluid, and even breast milk. 36ody fluid-derived exosomes are a highly stable reservoir of disease biomarkers, assisting liquid biopsy in various clinical settings such as cancers, cardiovascular diseases, and perinatal disorders. 37,38However, the coexisting contents and availability of each type of body fluid might create challenges to exosome isolation.
Production, isolation and purification of exosomes One of the major obstacles preventing exosome-based therapeutics from entering clinical practice is the low yield and efficiency of Table 1.The comparison between stem cell therapy and stem cell-derived exosome therapy

Treatment modality Advantages Limitations
Stem cell therapy multilineage differentiation potential short-lived viability and low engraftment after injection applicable to the treatment for a wide range of diseases stringent storage and transport requirements extensive accumulation of laboratory and clinical data tumorigenic potential easy to isolate and possible for mass-production infusion toxicity well-developed regulatory guidelines immunogenicity ethical issues Stem cell-derived exosome therapy comparable therapeutic effects to stem cells but much smaller batch-to-batch inconsistency more concentrated functional cargos, e.g., cytokines no standardized protocol for purification and storage modifiable at its surface and in its cargos relatively low yield for large scale manufacturing versatile delivery modalities no industry-standard quality specifications stable for long-term storage and transport insufficient regulatory control negligible risk of tumorigenesis and immune response lack of ethical issues Clinical applications of stem cell-derived exosomes Tan et al.
exosomes.For example, only less than 1 μg exosomal protein could be harvested from 1 ml culture medium in a laboratory setting. 39There are various methods of upscaling exosome production, which are categorized into biochemical strategies (e.g., LPS, BMP-2, HIF-1α, and IFN-γ and TNF-α), physical strategies (hypoxia, thermal stress, and starvation), mechanical strategies (shear stress and 3D culturing) and instrumental strategies (hollow-fiber bioreactors and stirred tank bioreactors). 40xosomes are heterogeneous in terms of size, content, surface markers, and source, which makes their isolation difficult.The currently available techniques for exosome isolation and purification are based on their size, surface charge, or immunoaffinity 26 (Fig. 1a).However, there is no 'one-fits-all' approach as these techniques all have advantages and disadvantages.
For example, ultracentrifugation is deemed the gold standard for exosome extraction.Although it requires minimal reagents and expertize, the time consumption, high cost, low efficiency, and lipoprotein co-separation have limited its large-scale use. 41mmunoaffinity chromatography is a separation technology based on the specific binding of antibodies and ligands.It is rapid and provides high purity, specificity, and yield.However, the antigen/ protein coupling used needs to be expressed on the surface of exosomes. 19Size-based isolation techniques mainly refer to ultrafiltration and size-exclusion chromatography, both of which Fig. 2 Illustration of the downstream surgical applications of exosome therapy (figure generated using Adobe Photoshop 2023 and Adobe Illustrator 2023).The therapeutic effects of exosomes are a hierarchical translation through disease-specific tissue responses, tissue-specific cellular alterations, and cell-specific molecular signaling pathways are quick and suitable for large-scale applications.But pore clogging, exosome loss, and low purity are making this method difficult to popularize. 42Although no single technique is perfect, combining the above techniques with others (e.g., precipitationbased and microfluidics-based) might be a solution to simultaneously meet multiple requirements for exosome isolation and purification.

Modification of exosomes
Exosomes can be biochemically modified to broaden, change, or improve their therapeutic effects.The modification of exosomes is classified into internal strategies (e.g., drug loading) and external strategies (e.g., surface modification).On the one hand, exosomes may be an ideal therapeutic carrier to deliver drugs, nucleic acids, and vaccines due to their advantages in stability, non-immunogenicity, and targeting recipient cells. 43There are various cargo loading techniques including pre-production loading methods (e.g., transfection, co-incubation, and electroporation) and postproduction loading methods (e.g., freeze-thaw cycles, incubation, sonication, extrusion, and hypotonic dialysis) depending on whether they are applied before or after exosome biogenesis 10,26,[44][45][46] (Fig. 1c).For example, Tian et al. loaded doxorubicin in Dex using electroporation for the treatment of breast cancer. 47im et al. loaded paclitaxel in RAW 264.7-derived exosomes using incubation and sonication to overcome multidrug resistance in cancer cells. 48Ohno et al. loaded antitumor let-7a miRNA in HEK293-derived exosomes using transfection to manage breast cancer. 49n the other hand, surface modification of exosomes is exemplified by genetic engineering of exosomal membrane or parental cells, chemical connection of targeting ligands, electrostatic interaction, and magnetic nanoparticle technology. 10The main purpose of surface modification is to selectively deliver exosomes to target cells for precise treatment.For example, Alvarez-Erviti et al. modified DCs using genetic engineering to express Lamp2b and RVG peptides, thereby targeting the central nervous system (CNS). 50Zhu et al. inserted tumor-targeting peptides, c(RGDyK), into the exosome surface using a chemical reaction to target glioblastoma. 51Nakase et al. bound exosomes with a complex formed by pH-sensitive fusion peptide and cationic lipid using electrostatic interaction, thereby achieving enhanced cytosolic delivery. 52aracterization and verification of exosomes Exosomes need to undergo characterization and verification before therapeutic applications.Current methods used for exosome characterization mainly focus on the size, morphology, and cargo profile of exosomes. 43Size-oriented verification includes nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and tunable resistive pulse sensing (TRPS), whereas morphology-oriented analysis includes scanning electron microscopy (SEM) and transmission electron microscopy (TEM). 19n addition, cargo profiling is further subdivided into proteomic, lipidomic, and genomic analyses including western blotting, ELISA, flow cytometry, mass spectroscopy, and PCR. 36Since each of the above characterization methods has advantages and disadvantages, it is a universal practice to combine analyses from three different aspects, e.g., a package of TEM, NTA, and western blotting, to identify isolated exosomes.
For example, microscopy-based methods, such as SEM and TEM, can directly visualize the surface topography and internal structure, respectively.However, TEM is not suitable for quick measurement of a large number of samples due to complicated operation and tedious sample preparation. 53NTA facilitates fast detection and real-time exosome observation while having a higher resolution than flow cytometry.The main disadvantage of NTA is its difficulty in distinguishing exosomes from contaminated proteins. 54As a mature technique, western blotting can qualitatively and quantitatively detect the expression of exosomal protein biomarkers, especially exosomes from cell culture media.However, it is time-consuming and not suitable for the detection of exosomes from biological fluids. 55,56orage of exosomes The currently used preservation methods for long-term storage of exosomes mainly include cryopreservation, lyophilization, and spray-drying. 10Temperature and antifreeze are the two most important ingredients for cryopreservation.Storage at 4 °C might weaken the biological activity and reduce the protein cargo of exosomes, whereas −80 °C is considered the optimal temperature causing the least impact on exosome morphology and content. 57,58Non-permeable disaccharide antifreeze, especially trehalose, represents the best choice as it prevents exosome aggregation and cryodamage. 59Heat-sensitive materials, e.g., exosomes and vaccines, treated by lyophilization of freezedrying can be easily stored and reconstituted by simply adding water.A recent study showed that lyophilization with cryoprotectant could retain the activity of exosomal proteins and RNA for approximately 4 weeks even when stored at room temperature. 60inally, in contrast to freeze-drying, spray-drying is a single-step process, thereby reducing the need for expensive equipment and lengthy multi-step milling.However, core parameters of spraydrying such as exosome feeding rate, atomization pressure, and outlet temperature, can all affect exosome stability and cargo integrity. 61THOPEDIC AND TRAUMA SURGERY AND SC-EXO THERAPY Fracture Fractures are the most common traumatic large-organ injuries, and approximately 10% heal improperly. 62Fracture healing involves an anabolic tissue-bulking phase and a catabolic tissueremodeling phase, which are controlled by various factors such as stem cells, innate and adaptive immune functions, and stability. 63iopharmacological treatment for fractures can be given locally (e.g., bone morphogenetic protein, BMP) or systemically (e.g., parathyroid hormone, PTH).As a promising alternative, exosome therapy for fracture healing mostly utilizes bone marrow-derived MSCs as a cellular supplier (Table 2).
The presumed mechanism of how MSC-derived exosomes promote fracture healing is as follows.Firstly, the progression of bone repair needs a variety of cells, e.g., inflammatory cells in the inflammation stage, endothelial and mesenchymal progenitor cells in the fibrovascular stage, osteoblasts and chondrocytes during bone formation, and osteoclasts during callus remodeling. 62Secondly, most of these cells can uptake exosomes, especially osteoblasts and vascular endothelial cells, 64 which are most related to fracture healing.Lastly, upon exosome absorption, the gene expression of the recipient cells is modified, thereby activating various signaling pathways (Fig. 3a), causing various cellular and tissue responses (Fig. 3b) and ultimately leading to improved fracture healing.
Early research has employed various animal models of fracture healing.In a transverse femoral shaft fracture model, exosomes were found to not only promote osteogenesis in wild-type mice, but also rescue retardation of fracture healing in CD9 − / − mice, a strain known to have a lower bone union rate. 65In a femoral nonunion model, exosomes enhanced fracture healing by promoting osteogenesis and angiogenesis possibly via the BMP-2/Smad1/RUNX2 pathway. 66In a tibial distraction osteogenesis model, exosomes secreted by young MSCs promoted osteogenic capacity of older MSCs and enhanced new bone formation in older rats. 67In addition, EPC-derived exosomes accelerated bone regeneration during distraction osteogenesis by stimulating angiogenesis. 68 As a major cargo of exosomes (Section 2.1), RNA can alter recipient cell gene expression and phenotypic function, with microRNA (miRNA) and long non-coding RNA (lncRNA) being the most widely studied. 69From the perspective of an exosomal miRNA, one group discovered that miR-136-5p from bone marrow MSC-derived exosomes promoted osteoblast proliferation and differentiation in vitro, thereby promoting fracture healing in vivo. 70This was achieved by inhibiting the downstream target gene of miR-163-5p, low-density lipoprotein receptor-related protein 4 (LRP4), through the Wnt/β-catenin pathway.The other group found that MSC-derived exosomal miR-25 could regulate the ubiquitination and degradation of Runt-related transcription factor 2 (Runx2) by Smad ubiquitination regulatory factor 1 (SMURF1) to promote fracture healing in mice. 71From a lncRNA perspective, especially the bone-specific lncRNA H19, a Chinese group revealed that although a high-fat diet reduced osteogenic differentiation and weakened fracture healing, this could be reversed by MSC-derived exosomal lncRNA H19 via miR-467/ HoxA10 axis in an obesity-induced fracture model. 72In addition, an American group demonstrated that exosomal lncRNA H19 not only improved osteogenesis but also angiogenesis through the angiopoietin 1/Tie2-NO signaling pathway in an immunocompromised nude mouse model. 73nstead of using naturally derived exosomes from MSCs, some researchers have conducted pre-isolation modification of exosomes to achieve better results.Liang et al. preconditioned MSCs with low doses of dimethyloxaloylglycine (DMOG), a small angiogenic molecule, to prepare the exosomes for an enhanced angiogenesis and bone regeneration in a critical-sized calvarial defect model by targeting the protein kinase B/mechanistic target of rapamycin (Akt/mTOR) pathway. 74Alternatively, Lu et al. loaded MSC-derived exosomes with miR-29a, which showed a robust ability in promoting angiogenesis and osteogenesis by targeting vasohibin 1. 75 Furthermore, umbilical cord MSC-derived exosomes demonstrated comparable results to their bone marrow counterparts during fracture healing. 76In addition, exosomes derived from MSCs under hypoxia exhibited better effects on bone fracture healing than those under normoxia.Mechanistically, hypoxia preconditioning enhanced the production of exosomal miR-126 through the activation of hypoxia-inducible factor 1 (HIF-1α).Various studies have shown that hypoxia preconditioning represents an effective and promising optimization of the therapeutic effects of MSC-derived exosomes for bone fracture healing.

Osteoarthritis
Osteoarthritis (OA) is the most common joint disease and most frequent reason for activity limitation in adults, affecting approximately 240 million patients globally. 77The pathology of OA has evolved from being viewed as cartilage-only to a multitissue disease that affects all components of the whole joint, including bone, synovium, muscle, ligament, and periarticular fat. 78Clinical trials have successfully revealed systemic compounds that arrest structural progression (e.g., cathepsin K and Wnt inhibitors) or reduce OA pain (e.g., nerve growth factor inhibitors).As a potential treatment option for OA, most MSCderived exosome therapy used chondrocytes as a target in in vitro models.These MSCs could originate from various tissues, such as bone marrow, synovium, gingiva, and infrapatellar fat pads (IPFPs).
Some studies focusing on chondrogenesis demonstrated a particular interest in the role of miRNA.Wu et al. found that IPFP MSC-derived exosomes protect articular cartilage from damage and ameliorate gait abnormality in OA mice by miR100-5pregulated inhibition of mTOR-autophagy pathway. 79Since it is easy to retrieve human IPFP from OA patients by arthroscopic operation within a clinic, this type of exosome therapy might simplify and accelerate the process from bench to bedside.Liu et al. discovered that MSC-derived exosomes could promote proliferation and inhibit apoptosis of chondrocytes via lncRNA-KLF3-AS1/miR-206/GIT1 axis in OA. 80 The cellular work conducted by Kong et al. showed that synovial MSC-derived exosomal miR-320c could enhance chondrogenesis by targeting ADAM19. 81In addition, Mao et al. suggested that exosomal miR-92a-3p from chondrogenic MSCs could enhance chondrogenesis and suppress cartilage degradation via targeting Wnt5a. 82In contrast to these studies using original exosomes, few groups modified exosomes prior to their systemic administration.Tao et al. modified exosomes by transfecting synovial MSCs with miR-140-5p and found that exosomal miR-140-5p-overexpression could enhance cartilage tissue regeneration and prevent OA of the knee in a rat model. 83Meanwhile, Wang et al. used TGF-β1 to stimulate MSCs, and the resultant exosomal miR-135b increased chondrocyte proliferation by regulating specificity protein-1. 84In a comparative study, Zhu et al. demonstrated that exosomes from iPSC-derived MSCs could provide a stronger therapeutic effect on OA than synovial membrane MSC-derived exosomes. 85ther studies have focused on not only chondrogenesis but also anti-inflammation and immune modulation during OA treatment.For example, MSC-derived exosomes inhibited inflammatory factors, glutamine metabolic activity-related proteins, glutamine, and GSH/GSSG ratio in vitro, while improving mice's chondrocyte function, tissue inflammation, and exercise capacity in vivo, thereby alleviating OA progression. 86Using a holistic approach, recent studies have shifted the attention away from cartilage towards other tissues (e.g., bone) in a diarthrodial joint.Firstly, bone marrow MSC-derived exosomal miR-206 promoted proliferation and osteogenic differentiation of osteoblasts in OA by reducing E74-like factor 3 (Elf 3), and ameliorated inflammation and increased expression of osteocalcin and BMP2 in mouse femoral tissues. 87Secondly, MSC exosome-treated osteochondral defects demonstrated a regenerative immune phenotype, characterized by a higher infiltration of CD163 + M2 macrophages over CD86 + M1 macrophages, with a concomitant reduction in proinflammatory synovial cytokines IL-1β and TNF-α. 88Lastly, gingival MSC-derived exosomes proved to be immunosuppressive in preventing collagen-induced arthritis. 89Compared with parental cells, these exosomes had the same or stronger effects in inhibiting IL-17A and promoting IL-10, reducing incidences and bone erosion by arthritis, via inhibiting the IL-17RA-Act1-TRAF6-NF-κB signaling pathway.
Currently, there is no single 'one size fits all' drug that may be suitable for all OA patients.Disease-modifying OA drugs (DMOADs) might become the next-generation OA treatment. 90It is very valuable and relevant that MSC-derived exosome therapy for OA coincides with DMOADs: both are capable of targeting inflammatory cytokines, matrix-degrading enzymes, and the Wnt pathway.Thus, emerging approaches for DMOAD development, such as miRNA-based modality and targeting cellular senescence, might also be used to refine MSC-based exosome therapy for OA.

Spinal cord injury
Traumatic spinal cord injury (SCI) is a devastating global health issue that poses a significant functional and economic burden both on the patient and society. 91The pathophysiology of SCI includes primary injuries caused by mechanical trauma and secondary injury cascade characterized by apoptosis, edema, ischemia, inflammatory cell infiltration, and excitotoxicity. 92espite surgical intervention, clinical studies involving pharmacotherapy can be broadly classified as either neuroprotective or neuroregenerative. 93Targeting each event of the above mechanistic chain, both MSC-and NSC-derived exosome therapy could exert a beneficial influence on spinal cord protection and regeneration.
Some groups have targeted neuronal cell death.Ma et al. revealed that insulin-like growth factor 1 (IGF-1)-stimulated NSCderived exosomes could inhibit neuronal apoptosis while promoting functional recovery after SCI through a miR-219a-2-3p/ YY1 pathway. 94Alternatively, Zhang et al. discovered that subarachnoid injection of NSC-derived exosomes could suppress neuronal cell apoptosis by activating autophagy via miR-374-5p/ STK-4 axis for enhanced functional recovery in SCI. 95Shao et al. explored other forms of cell death (e.g., ferroptosis) using MSCderived exosomes, and found that exosomal lncGm36569 could inhibit neuronal cell ferroptosis via miR-5627-5p/FSP1 axis, thereby decreasing neuronal dysfunction. 96ome groups have targeted anti-inflammation and immunomodulation.Nakazaki et al. discovered that fractionated intravenous infusion of MSC-derived exosomes could target M2 macrophages and upregulate TGF-β, thereby stabilizing microvessels and improving functional recovery. 97Similarly, Liu et al. demonstrated that in addition to hypoxia increasing exosome production from bone marrow MSCs, preconditioned exosomal miR-216a-5p could also repair traumatic SCI by shifting microglial M1/M2 polarization via the TLR4/NF-κB/PI3K/Akt pathway. 98uang et al. valuably proved that epidural fat MSC-derived exosomes could attenuate NLRP3 inflammasome and improve functional recovery in SCI. 99Compared to MSC-derived exosomes, exosomes derived from EPCs could provide comparable antiinflammatory effect.Yuan et al. showed that the exosomal miR-222-3p from EPCs could promote anti-inflammatory macrophages via the SOC3/JAK2/STAT3 pathway and improve mouse functional repair after SCI. 100 Some groups have targeted angiogenesis and blood-spinal cord barrier (BSCB) integrity.For example, Zhong et al. used unmodified NSC-derived exosomes, found that they were highly enriched in VEGF-A, and could therefore enhance the angiogenic activity of spinal cord microvascular endothelial cells (SCMECs). 101In comparison, Chen and co-workers modified NSC-derived exosomes with FTY720, an immune modulator and microvascular regulator, to protect the barrier function of SCMECs via the PTEN/Akt pathway, thereby ameliorating hindlimb function. 102It is well-known that the connection between the microvascular endothelium of the spinal cord and the pericyte is crucial in maintaining the structural integrity of BSCB. 103Thus, Zhou's team attempted to verify the role of exosome therapy in pericyte homeostasis. 104They proved that bone marrow MSC-derived exosomes could reduce pericyte pyroptosis and increase pericyte survival rate in vitro, while improving BSCB integrity and locomotor recovery in vivo.
Finally, some groups have targeted other aspects during neuroprotection and neuroregeneration, such as neurotoxic astrocytes and endogenous NSC sustainability.Lai et al. proved that human umbilical cord MSC-derived exosomes could facilitate recovery of spinal cord function by targeting neurotoxic astrocytes. 105In addition, miR-146a-5p-modified exosomes exerted a more powerful effect than unmodified exosomes.Li et al. discovered that exosomes derived from nerve growth factor (NGF)-overexpressing bone marrow MSCs could enhance neuronal differentiation of NSCs and axonal regeneration. 106Zhou et al. demonstrated that placental MSC-derived exosomes could promote the activation of proliferating endogenous NSCs, thereby improving both locomotor activity and bladder dysfunction, 107 which is a frequent sequelae that could further worsen the quality of life of SCI patients. 108scle and tendon tear Muscle and tendon tears can result from either acute trauma (e.g., fractures, Section 3.1) or chronic overuse (e.g., sports injury).109 Healing of muscle strain and tendon tear follows the typical wound healing course, involving the inflammatory, proliferative, and remodeling phases.Multiple non-surgical strategies have been trialed to improve healing, including cell-based and growth factor-based therapies.110 The following proof-of-concept studies indicate that MSC-derived exosomes could become the nextgeneration musculoskeletal treatment.
On the one hand, some groups have focused on individual components of the muscle-tendon-bone unit.Nakamura et al. claimed that MSC-derived exosomes could improve in vitro myogenesis in C2C12 myoblasts and angiogenesis in HUVECs, while accelerating in vivo skeletal muscle regeneration in a cardiotoxin-induced muscle injury model. 111These benefits were at least in part mediated by miRNAs such as miR-494.Chen et al. discovered that exosomes from adipose MSCs could enhance the proliferation and the migration of primary tenocytes, while also improving mechanical strength of repaired tendons by upregulating decorin and biglycan in a rabbit Achilles tendon rupture model. 112n the other hand, some groups have regarded the muscletendon-bone unit as a single functional system and used rotator cuff tear as the disease model, which is the most common shoulder condition for which patients seek treatment. 113One group of researchers published two consecutive studies using adipose MSC-derived exosomes.In a rat model of massive rotator cuff tear, exosome therapy could prevent the atrophy, inflammation, and vascularization of muscles. 114In a rabbit model of chronic rotator cuff tear, exosome therapy could prevent fatty infiltration and improve biomechanical properties. 115Another group reported that bone marrow MSC-derived exosomes could increase the breaking load and stiffness of the rotator cuff after reconstruction, induce angiogenesis around the rotator cuff endpoint, and promote growth of the tendon-bone interface. 116her orthopedic diseases Osteoporosis is a metabolic bone disease characterized by low bone density and weakening of bone architecture, which increase the risk of fractures.It results from osteoclastic bone resorption undercompensated by osteoblastic bone formation. 117In a cellular study, adipose MSC-derived exosomes could antagonize hypoxia/ serum deprivation-induced osteocyte apoptosis and osteocytemediated osteoclastogenesis. 118Further animal studies revealed that umbilical cord MSC-derived exosomes could inhibit bone marrow MSC apoptosis and prevent disuse osteoporosis via miR-1263/Mob1/Hippo pathway, 119 and improve tibial density and reverse estrogen-deficient osteoporosis via miR-2110 and miR-328-3p. 120ompared to SCI, damage to peripheral nerve (e.g., sciatic nerve injury) is considerably more common.The subsequent nerve regeneration is controlled by the interplay between neurons and Schwann cells, and further complicated by inflammatory cell infiltration. 121It was shown that adipose MSC-derived exosomes could target neurons by increasing neurite outgrowth in vitro and axonal regeneration and walking behavior in vivo. 122Adipose MSC-derived exosomes could target Schwann cells by promoting the proliferation, migration and secretion of neurotrophic factors in vitro and restore denervation muscle atrophy in vivo. 123LPSpreconditioned MSC-derived exosomes could target inflammatory cells by enhancing M2 macrophage polarization in vitro and accelerate peripheral nerve regeneration in vivo. 124ntervertebral disc (IVD) degeneration is a major cause of lower back pain which is the leading injury in total global years lived with disability.Its molecular processes include extracellular matrix (ECM) degeneration, inflammation, oxidative stress, apoptosis, senescence and reduced autophagy. 125The emerging avenues of exosome therapy attempt to solve some of these issues.Cheng et al. demonstrated that intradiscal injection of bone marrow MSCderived exosomes could inhibit nucleus pulposus cell (NPC) apoptosis and alleviate IVD degeneration via exosomal miR-21. 126n the other hand, Chen et al. discovered that human ESC-derived exosomes could inhibit NLRP3 inflammasome to alleviate pyroptosis in nucleus pulposus cells by delivering miR-302c. 127n addition to cell death and mitochondrial damage, oxidative stress in NPCs was also found to be inhibited by MSC-derived exosomes. 128Since IVD degeneration and OA share a common molecular disease spectrum 125 the positive results of OA treatment using MSC-derived exosomes (Section 3.2) could be used as a reference for IVD degeneration research.
Osteonecrosis, aka., avascular necrosis, of the femoral head (ONFH) is a disabling condition affecting a younger population, which often results in total hip arthroplasty. 129Glucocorticoid (GC)-induced osteonecrosis is one of the most common causes of ONFH, whose pathogenesis is manifested in two aspects: compromised blood supply to the femoral head and dampened osteogenic activity.Liu et al. showed that exosomes from iPSCderived MSCs could prevent GC-induced ONFH by promoting angiogenesis and osteogenesis via the PI3K/Akt pathway. 130Zuo et al. demonstrated that miR-26a-overexpressing exosomes derived from HSCs could provide similar therapeutic effects. 131UROSURGERY AND SC-EXO THERAPY Ischemic stroke Strokes are the second highest cause of death and the third leading cause of disability globally, with ischemic stroke being the most common subtype. 132The key events during the ischemic cascade include neuronal dysfunction, excitotoxicity, neurochemical injury, and neuroinflammation. 133In terms of treatment, a new generation of clinical trials is now underway, which uses cytoprotective drugs, such as immunomodulators, IL-6 receptor antagonists, Rho kinase inhibitors, and free radical scavengers. 134argeting each event of the above pathophysiology, nearly all subtypes of SC-exo demonstrated potent therapeutic effects on stroke recovery (Table 3).Some groups have targeted neuroprotection and neurogenesis.Firstly, SC-exo therapy could inhibit neuronal cell death.Luo et al. found that NSC-derived exosomes could inhibit apoptosis while promoting the proliferation of SH-SY5Y cells both under normal and oxygen-glucose deprivation (OGD) conditions. 135This was also tested in a middle cerebral artery occlusion (MCAO) model as a reduced infarction area and neuronal apoptosis via exosomal miR-150-3p.Other in vitro and in vivo studies showed similar antiapoptotic effects using EPC-derived exosomes. 136,137Zhang et al. discovered that the exosomal anti-apoptotic effect could be improved by preconditioning the parental NSCs with interferon gamma (IFN-γ). 138Secondly, SC-exo therapy could protect cells of the CNS.Kang et al. revealed that exosomes derived from bone marrow MSCs could rescue OGD-induced injury in neural cells by suppressing NLRP3 inflammasome-mediated pyroptosis. 139Exosomes sourced from hypoxic cultures had a more pronounced neuroprotective effect than their counterparts from normal cultures.Similarly, Li et al. discovered that exosomes derived from human iPSC-derived neural progenitor cells exhibited a neuroprotective effect on OGD neurons and neurite outgrowth. 140his protection of neuronal function under ischemic conditions was regulated through the PTEN/Akt pathway.In addition, Sun et al. proved that NSC-derived exosomes could also protect astrocytes, which become supporting reactive astrocytes (RAs) after strokes. 141Thirdly, SC-exo therapy could improve post-stroke neurogenesis.Wei et al. suggested that Zeb2/Axin2 from bone marrow MSC-derived exosomes could improve post-stroke neurogenesis, neural plasticity, and spatial memory and nerve function, likely via the SOX10, Wnt/β-catenin, and endothelin-3/ EDNRB pathways. 142Wang et al. illustrated that miR-126-modified EPC-derived exosomes could alleviate acute brain injury and promote functional recovery after stroke by enhancing neurogenesis. 143ome groups have targeted the inhibition of the neuroinflammation.Firstly, unmodified SC-exo therapy exhibited an antiinflammatory effect through exosomal miRNAs.Dong et al. showed that bone marrow MSC-derived exosomes could induce BV2 microglia deactivation and M2 polarization in vitro, while reducing infarct size and improving neuronal function in vivo via transferring miR-23a-3p. 144Similarly, Zhang et al. unveiled that umbilical cord MSC-derived exosomal miR-146a-5p could attenuate microglia-mediated neuroinflammation after OGD in vitro, while improving behavioral deficits and microglia activation in vivo via the IRAK1/TRAF6 signaling pathway. 145Secondly, the anti-inflammatory effect of SC-exo therapy could be enhanced by modifying the exosomes.Yoon and co-workers established tumor susceptibility gene (TSG)101-overexpressing human NSCs, thereby increasing exosome secretion. 146The engineered exosomes not only attenuated LDH release and proinflammatory factors in vitro, but also reduced infarction volume, inhibited DNA-damage pathway, and upregulated neurotrophic factors in vivo.Furthermore, Tian's team broke new ground by ingeniously attaching RGD peptide onto an NSC-derived exosome membrane, which targeted the lesion region of the ischemic brain after intravenous administration, thereby suppressing the inflammatory response after cerebral ischemia by inhibiting the MAPK pathway. 147nterestingly, Gao et al. used induced NSCs (iNSCs) reprogrammed from mouse fibroblasts for stroke treatment.They showed that iNSC-derived exosomes, bearing similar therapeutic effects with NSC-derived ones, could not only promote neurogenesis but also inhibit neuroinflammation. 148inally, some groups have targeted other aspects during stroke recovery, such as neurochemical injury and oxidative stress.Zhu et al. loaded brain-derived neurotrophic factor (BDNF) into exosomes derived from NSCs to construct engineered exosomes. 149In a model of H 2 O 2 -induced oxidative stress, exosome therapy significantly enhanced NSC survival.In a rat MCAO model, exosome therapy not only inhibited microglial activation, but also boosted the differentiation of endogenous NSCs into neurons.Collectively, BDNF-based modification of NSC-derived exosomes has improved effects in the treatment of ischemic stroke.On the other hand, miR-210-modified EPC-derived exosomes could protect neurons from hypoxia and reoxygenation (H/R)-induced apoptosis, oxidative stress, and decreased viability, thereby supporting the treatment of ischemic stroke. 150,151The exosomal miR-17-5p from ACE2-enriched EPC-derived exosomes could ameliorate cerebral ischemic injury in aged mice. 152In an intriguing study conducted by Xu and co-workers, combination of NSC-exo and EPC-exo with miR-210 and miR-123 overexpression exerted better therapeutic effects on ischemic stroke by protecting H/R injured neurons through the BDNF-TrkB and Nox2/ ROS pathways. 153n contrast to ischemic stroke, hemorrhagic stroke poses a deadlier threat and worse disability in most survivors. 154miR-137 overexpression was found to boost the neuroprotective effects of EPC-derived exosomes against apoptosis, ferroptosis, and mitochondrial dysfunction in oxyhemoglobin-treated SH-SY5Y cells, an in vitro hemorrhagic stroke model, partially through the COX2/ PGE2 pathway. 155aumatic brain injury Approximately 70 million patients suffer from traumatic brain injury (TBI) globally each year, which poses serious physical, psychosocial and economic threats. 156TBI can be categorized as primary injuries (e.g., axonal death, neuroinflammation, neurochemical change, and metabolic dysfunction) and secondary injuries (e.g., ischemic and hypoxic damage, cerebral edema, raised intracranial pressure, hydrocephalus, and infection). 157Each patient with a TBI has a unique set of circumstances depending on variables such as the location and severity of the injury, making medical and surgical treatment quite challenging. 158Therefore, systemic therapy using SC-exo may become a 'one-size-fits-all' option for managing TBI.
A series of animal studies published initially focused on the functional recovery and macroscopic aspects of MSC-derived exosome therapy.In a rat TBI model, exosome-treated animals showed significant improvement in spatial learning and sensorimotor function. 159In addition, exosome treatment significantly increased the number of newborn endothelial cells in the lesion boundary zone, and newborn immature and mature neurons in the dentate gyrus.In another rat TBI model with similar findings, exosomes derived from MSCs cultured in a 3D system provided better outcomes than those in a conventional 2D condition. 160In a monkey model of TBI to the primary motor cortex, exosome-treated animals returned to pre-operative grasp patterns and latency to retrieve a food reward in the first 3-5 weeks of recovery. 161In an even more complicated and clinically realistic large animal model, in which both TBI and hemorrhagic shock were investigated, exosome therapy attenuated the severity of neurologic injury and enabled faster neurologic recovery. 162n comparison, studies completed in recent years shed new light on the molecular mechanisms underlying SC-exo therapy for TBI.Chen et al. reported that adipose MSC-derived exosomes could promote functional recovery, suppress neuroinflammation, reduce neuronal apoptosis, and increase neurogenesis.This was achieved through the uptake of exosomes specifically by microglia and suppression of their activation by inhibiting the NF-κB & MAPK pathways. 163Wen et al. showed that bone marrow MSCderived exosomes could reduce cell apoptosis in cortical tissue of mouse models of TBI, inhibit neuroinflammation, and promote the transformation of microglia to the anti-inflammatory phenotype.This was realized by the action of miR-181b on the IL-10/STAT3 pathway. 164Abedi et al. proved that NSC-derived exosomes could improve neurobehavioral performance, inhibit astrocyte neuroinflammation, enhance neurogenesis, while maintaining NSC stemness. 165A valuable additional finding was that exosomes seemed to be superior to the parent NSCs in terms of sensorimotor functional recovery.Finally, a dose-response and therapeutic window demonstrated that MSC-derived exosomes could improve angiogenesis and neurogenesis, and sensorimotor and cognitive function, while reducing neuroinflammation and hippocampal neuronal cell loss. 166Although 100 µg and 1 day might be the optimal dose and therapeutic window respectively, exosomes exhibited a wide range of effective doses for treatment of TBI within a therapeutic window of at least 7 days post-injury.
TBI and SCI are two of the most severe CNS traumas, which are increasingly recognized as global health priorities.The emerging evidence presented in Sections 3.3 and 4.2 are mutually beneficial for these two closely related research subspecialties.Henceforth, future research on SC-exo therapy for TBI and SCI could be either mechanism-based (e.g., the role of brain-gut axis 167 transcriptional factors 168 inflammasome 169 and the complement system 170 ) or modification-based (loading exosomes with drugs, e.g., immunomodulators 171 antioxidants 172 circular RNAs 173 and microRNAs 174 ).
Alzheimer's disease Unlike TBI and SCI, which are traumatic in nature, Alzheimer's disease (AD) and Parkinson's disease (PD) are the most common neurodegenerative diseases (NDD).The hallmarks of NDD include, but are not limited to, pathological protein aggregation, synaptic and neuronal network dysfunction, aberrant proteostasis, cytoskeletal abnormalities, altered energy homeostasis, DNA and RNA defects, inflammation, and neuronal cell death. 175AD is the most common form of dementia globally and accounts for 25 million cases. 176Currently, only two classes of drugs are approved for symptomatic AD treatment, including cholinesterase inhibitors and NMDA antagonists.Although several therapeutics are actively undergoing clinical trials, none of them are near curative for AD. 177The challenges of brain-drug delivery, e.g., the bloodbrain barrier (BBB) and pharmacokinetic drawbacks, are very likely to be solved by nanosized exosomes, which are additionally packaged with potent biomolecules.Most SC-exo therapy involves amyloid-β (Aβ), which is positioned at the center of AD pathophysiology. 178e initial work focused on the clearance of aggregation of the pathological protein, Aβ peptide.The intracerebral injection of MSC-derived exosomes by Elia and co-workers reduced Aβ plaque burden and dystrophic neurites in both the cortex and hippocampus in the early stages of a preclinical model of AD. 179 In addition, using immunoblotting, the authors confirmed the presence of Neprilysin, a neutral endopeptidase capable of Aβ degradation, in the exosome's lysates and its mRNA.Some teams have focused on relieving synaptic dysfunction and oxidative stress.Wang et al. found that MSC-derived exosomes could rescue synaptic impairment and improve cognitive behavior in APP/PS1 mice, while alleviating exogenous Aβ-induced inducible nitric oxide synthase (iNOS) expression. 180nstead of using MSC-derived exosomes, Li et al. administered NSC-derived exosomes and enhanced mitochondrial function, sirtuin 1 activation, synaptic activity, and rescued cognitive deficits. 181Using alternative methods, Huber et al. noticed that heat shock-induced exosomes derived from NSCs exhibited greater neuroprotection against oxidative stress as well as Aβ-induced neurotoxicity. 182ome teams have centered their research around energy homeostasis.Chen et al. found that MSC-derived exosomes could improve brain glucose metabolism and cognitive function in AD transgenic mice using 18 F-FDG PET/CT imaging and NOR testing, respectively. 183ome teams have focused on microglial neuroinflammation.In Zavatti's cellular study, it was found that amniotic fluid MSCderived exosomes could mitigate neuroinflammatory microglial phenotype and recover neurotoxicity from Aβ using LPSstimulated BV2 microglia and SH-SY5Y neuroblastoma cells as models, respectively. 184Ding et al. showed that umbilical cord MSC-derived exosomes could alleviate neuroinflammation and reduce Aβ deposition by modulating microglial activation, thereby increasing spatial learning and memory function in AD mice. 185ome teams have focused on neuronal cell death and neurogenesis hoping to counteract AD progression.Reza-Zaldivar and co-workers gave MSC-derived exosomes to AD mice and the SC-exo therapy stimulated neurogenesis in the subventricular zone and alleviated Aβ-induced cognitive impairment. 186hese effects are comparable to those shown in the MSCs.
Some teams have focused on the BBB, the dysfunction of which leads to increased permeability, microbleeds, impaired glucose transport, and degeneration of pericytes and endothelial cells. 187iu et al. indicated that BBB breakdown in 5xFAD (familial Alzheimer's disease) mice occurred at 4 months of age, and more importantly, treatment with NSCs-derived exosomes reversed ADcaused BBB deficiency. 188inally, some groups have focused on improving the technicality of SC-exo therapy for AD.When exosomes are injected intravenously, they could be tracked in other organs instead of the targeted regions in the brain.Cui et al. conjugated MSC-derived exosomes with CNS-specific rabies viral glycoprotein (RVG) to target them to the brain of transgenic AD mice. 189The modified exosomes not only exhibited increased delivery to the cortex and hippocampus, but also significantly improved learning and memory capabilities with reduced Aβ deposition.On the other hand, Gao et al. obtained iNSCs through somatic cell reprogramming, which opened a new window for sourcing therapeutic exosomes.They demonstrated that iNSCderived exosomes, bearing comparable therapeutic effects with NSC-derived ones, could mitigate various AD phenotypes, e.g., cognitive function, Aβ deposition, neuroinflammation, and neuroregeneration, in a preclinical mouse model. 190rkinson's disease Parkinson's disease is the second most common neurodegenerative disease among the elderly, affecting more than 6 million patients worldwide.191 PD is caused by the necrosis of dopaminergic neurons in the substantia nigra and the presence of protein inclusions named Lewy bodies.The molecular pathophysiology includes α-synuclein proteostasis, mitochondrial dysfunction, oxidative stress, calcium imbalance, and neuroinflammation.192 In a study using bone marrow MSC-derived exosomes, Huang et al. discovered that exosome-derived TNF-stimulated gene-6 (TSG-6) could attenuate 1-methyl-4-phenylpyridinium ion (MPP + , metabolite of a neurotoxin MPTP)-induced neurotoxicity.In this in vitro PD model using SH-SY5Y and SK-N-SH cells, the exosomal anti-PD progression effect was found to be mediated through the STAT3/miR-7/NEDD4 axis. 193In a study using NSC-derived exosomes, Lee et al. revealed that SC-exo therapy could help to prevent the neuropathology and progression of PD. 194 Working in vitro on SH-SY5Y and BV2 cells, NSC-derived exosomes could reduce the intracellular reactive oxygen species (ROS) and associated apoptotic pathways.Working in vivo on 6-hydroxydopamine-induced PD mice, NSC-derived exosomes could downregulate pro-inflammatory factors and significantly reduce dopaminergic neuronal loss.The presence of NSC-specific microRNAs, such as miR-182-5p, miR-183-5p, miR-9 and let-7, was confirmed and found to be involved in cell differentiation, neurotrophic function, and immune modulation.

Multiple sclerosis
Multiple sclerosis (MS) is the most common non-traumatic, neurodegenerative, and disabling CNS disease affecting young adults.The pathological hallmark of MS is the formation of demyelinating lesions in the brain and spinal cord, with an inflammatory and autoimmune involvement. 195Currently licensed disease-modifying therapies include interferon-based, immunomodulatory, immunosuppressive, and immune reconstitution drugs. 196A few preliminary studies have highlighted the potential of MSC-derived exosomes for MS treatment.
In an animal experiment using experimental autoimmune encephalomyelitis (EAE) rats, Li et al. showed that SC-exo therapy significantly decreased neural behavioral scores, reduced the infiltration of inflammatory cells into the CNS, and decreased demyelination. 197In addition, exosome treatment upregulated M2-related cytokines while downregulating M1-related ones by regulating the polarization of microglia.
In an animal study using two mouse models of demyelination (the EAE model and the cuprizone diet model), Zhang et al. found that SC-exo therapy could promote remyelination by acting both directly on oligodendrocyte (OL) progenitors and indirectly on microglia. 198MSC-derived exosomes could improve neurological outcomes, increase the numbers of newly generated and mature OLs, decrease Aβ precursor protein density, decrease neuroinflammation by shifting from M1 to M2 phenotype, and inhibit the TLR2/IRAK1/NF-κB pathway.

Other neurosurgical and related diseases
The surgical potential of MSC-and NSC-derived exosome therapy in four major types of neurosurgical or neurological diseases has been thoroughly discussed above.In addition to vascular disruption-, trauma-, neurodegeneration-, and autoimmune-related disorders, other diseases have been proven suitable targets for SC-exo therapy recently (Table 3).These include, but are not limited to: 1, dementia, such as vascular dementia. 199HIV-associated neurocognitive disorders 200 and radiation-induced cognitive dysfunction 201,202 ; 2, functional disorders, such as epilepsy 203 and mechanical allodynia 204 ; 3, congenital abnormalities, such as spina bifida aperta 205 ; 4, neuropsychological conditions, such as depression 206 and stress 207 ; 5, brain aging [208][209][210] ; 6, iatrogenic brain problems, such as deep hypothermic circulatory arrest. 211ASTIC SURGERY AND SC-EXO THERAPY Wound healing occurs in all parts of the human body, with cutaneous wounds being the most common.The highest wound-related expenses were attributed to surgical wounds followed by diabetic ulcers. 212The overall but overlapping phases of wound healing include hemostasis, inflammation, angiogenesis, proliferation and remodeling, each of which is governed by distinct cell types and modulated by various signaling pathways. 213More than half of relevant work using SC-exo therapy to boost cutaneous wound healing is MSC-based (Table 4).
In the inflammatory stage, exosomes could inhibit the proliferation of peripheral blood mononuclear cells and promote the transformation of regulatory T cells in vitro, and reduce the number of lymphocytic infiltrations in the skin. 214In addition, exosomes could reduce IgE, eosinophil and mast cell count, and downregulate inflammatory cytokines. 2157][218] EPC-derived exosomes could accelerate cutaneous wound healing by promoting angiogenesis 219 through the Erk1/2 pathway 220 and p53 pathway. 221In the proliferative stage, stem cell-derived exosomes could promote the proliferation and migration of fibroblasts and keratinocytes.Some were achieved through the PI3K/Akt 222 Akt/HIF-1α 223 ERK1/2 224 and Wnt/β-catenin 225 pathways, while others through inhibition of LATS2 226 PPARγ 227 and AIF nucleus translocation 228 In the final remodeling stage of wound healing, granulation tissue is replaced by permanent scar, during which abnormal wound healing might occur (e.g., keloids and hypertrophic scars).MSC-derived exosomes could suppress fibroblast-myofibroblast transition via the TGF-β/Smad2 pathway 229,230 and increase collagen synthesis in early stage and reduce in late stage 231 thereby reducing scar formation.
Furthermore, ESC-derived exosomes were found to exert similar therapeutic effect for wound healing to MSC-derived ones.Chen et al. used human ESC-derived exosomes to help healing of pressure ulcer. 232They noticed that exosomes could ameliorate endothelial senescence by activating Nrf2 and recover agingrelated angiogenic dysfunction, thereby accelerating wound healing. 232In addition, Bae et al. revealed that the exosomal mmu-miR-291a-3p from ESCs could inhibit cellular senescence in human dermal fibroblasts through the TGF-β receptor 2 pathway, thereby accelerating the excisional skin wound healing process. 233n addition to wound healing, other plastic surgery-related diseases have been proven to be suitable targets for SC-exo therapy (Table 4).These include, but are not limited to: 1, skin grafting, such as skin flaps 234 ; 2, tissue loss, such as craniofacial defect 235 ; 3, autoimmune skin diseases, such as scleroderma 236 ; 4, skin infections, such as leishmaniasis 237 ; 5, hair transplantation, such as for alopecia 238 ; 6, skin aging 239

GENERAL SURGERY AND SC-EXO THERAPY
As a major subspecialty of general surgery, hepatobiliary surgery has attracted tremendous attention to SC-exo therapy.Firstly, acute liver injury (ALI)/acute liver failure (ALF) is a rare but challenging syndrome manifested by hepatic dysfunction, coagulopathy, encephalopathy, and multiorgan failure.About 60% of cases with ALF require and undergo orthotopic liver transplantation or result in death. 240In one study, Lin's team focused on the cell death aspect of ALI, and found that MSC-derived exosomes could protect against ferroptosis via stabilization of SLC7A11 in carbon tetrachloride-induced ALI. 241Alternatively, Shao's team focused on the pre-isolation modification of the exosomes, and revealed that exosomes derived from umbilical cord MSCs could ameliorate IL-6-induced ALI through exosomal miR-455-3p. 242econdly, in contrast to ALI, liver fibrosis occurs when the liver sustains a chronic injury, which may progress into cirrhosis, liver failure, hepatocellular carcinoma, and even death. 243 pathway in human liver fibrosis. 244In addition, Wang et al. found that exosomes derived from 3D human ESC spheroids could attenuate hepatic stellate cell activation and inhibit liver fibrosis through inactivation of the Smad pathway by exosomal miR-6766-3p. 245For those with end-stage liver fibrosis needing a liver transplant, liver ischemia reperfusion injury (IRI) is a serious complication for graft dysfunction and organ rejection. 246Yang et al. demonstrated that bone marrow MSC-derived exosomes could relieve hepatic IRI, reduce hepatocyte apoptosis, and decrease liver enzyme levels by enhancing autophagy. 247Du et al. showed that exosomes from iPSC-derived MSCs could protect liver against hepatic IRI via activating sphingosine kinase and the sphingosine-1-phosphate pathway. 248Thirdly, nonalcoholic fatty liver disease (NAFLD) is known to adversely affect stroke recovery.Using a type 2 diabetes mellitus mouse model, Venkat et al. demonstrated that HSC-derived exosomes could simultaneously reduce liver dysfunction and improve neurological and cognitive function. 249Lastly, acute pancreatitis is an unpredictable and potentially lethal disease, the prognosis of which mainly depends on whether it develops into multiple organ dysfunction syndrome. 250Chen et al. that exosomes from iPSC-derived MSCs could improve myocardial injury caused by severe acute pancreatitis through the Akt/Nrf2/HO-1 pathway. 251eripheral artery disease affects 200 million patients worldwide and, in its most severe stage, can cause critical limb ischemia, subjecting patients to increased risk of cardiovascular events, amputation and death. 252As a cell-free therapy, placenta MSCderived exosome infusion could enhance angiogenesis in a murine auricle ischemic injury model using laser Doppler blood flow analysis. 253Mechanistically, MSC-derived exosomes not only promote tube-like structure formation in vitro, but also mobilize endothelial cells into subcutaneous Matrigel plug in vivo, mainly through exosomal pro-angiogenic microRNAs, such as miR-30b. 254n addition, HSC-derived exosomes could repair ischemic hindlimb in mice by improving limb perfusion, capillary density, motor function and their amputation. 255This was most likely caused by internalization of exosomal miR-126-3p by endothelial cells relative to smooth muscle cells and fibroblasts.Human iPSCderived exosomes demonstrated similar neoangiogenic effect through the exosomal miR-199b-5p. 256On the other hand, endovascular re-canalization is increasingly being used to reestablish blood flow to ischemic areas and restore tissue loss or gangrene for patients with peripheral artery disease. 2579][260] In addition, Kong et al. demonstrated similar protective effect of EPC-derived exosomes against balloon injury by inhibiting neo-intimal hyperplasia. 261This was achieved through promotion of reendothelialization and suppression of restenosis rather than through the direct inhibition of proliferation and migration of smooth muscle cells.
McCulloh and his co-workers published an intriguing study on the SC-exo therapy for necrotizing enterocolitis (NEC) which has an overall mortality of over 30% for premature infants requiring surgery. 262The authors compared the therapeutic effect of exosomes derived from four different types of stem cells, i.e., amniotic fluid MSCs, bone marrow MSCs, amniotic fluid NSCs and neonatal enteric NSCs. 263When injected at a concentration of at least 4 × 10 8 , all types of SC-exo were shown to reduce the incidence and severity of experimental NEC as effectively as their parental stem cells.
Sepsis is a deadly and potentially preventable complication in general surgery, in which microvascular dysfunction leads to multi-organ failure and mortality. 264Using a murine sepsis model by cecal ligation and puncture (CLP), Zhou and co-workers demonstrated that EPC-derived exosomes could improve sepsis outcome. 265This was manifested by reduced lung and renal vascular leakage, improved organ function, and increased survival through the exosomal miR-126-5p and miR-126-3p.Similarly, Liu et al. exhibited protective effect of EPC-derived exosomes on sepsis-induced organ damage and immune suppression by the exosomal miR-382-3p through the IκBα/NF-κB pathway. 266RDIOTHORACIC SURGERY AND SC-EXO THERAPY The world's leading mortality is ischemic heart disease (IHD) which is primarily caused by obstructive coronary atherosclerosis.The rupture of an atherosclerotic plaque is the most common trigger of acute arterial thrombosis causing myocardial infarction (MI).Prolonged oxygen deprivation to the myocardium can lead to cardiomyocyte death.Although timely reperfusion is essential, myocardial IRI might occur, thus mitigating the beneficial effects of reperfusion.Despite modern coronary reperfusion, the mortality and morbidity associated with the development of heart failure as a consequence of acute MI remain substantial, highlighting the importance of next-generation cardioprotective therapies, such as SC-exo. 267he  269 Similarly, human iPSC-derived exosomes could improve recovery from MI without increasing the frequency of arrhythmogenic complications in a swine model. 270The work conducted by Wen et al. and Santoso et al. focused on the death of cardiomyocytes.MSC-derived exosomes could ameliorate cardiomyocyte apoptosis in hypoxic conditions through miR-144 by targeting the PTEN/Akt pathway 271 whereas iPSC-derived exosomes could regulate autophagy in hypoxic cardiomyocytes. 272oth Katsur et al. and Chen et al. focused on myocardial IRI.The former team found that exosomes derived from noncardiomyocyte-related cells, i.e., CTX0E03 NSCs, could reduce infarct size while delaying cardiomyocyte mitochondrial permeability transition pore opening through the JAK/STAT pathway. 273he latter team discovered that MSC-derived exosomal miR-143-3p could suppress myocardial IRI by regulating autophagy via the CHK2-Beclin2 pathway. 274Finally, Chen and co-workers proved that bone marrow MSC-derived exosomes could attenuate cardiac hypertrophy and fibrosis in pressure overload-induced remodeling, thereby providing a promising potential treatment for heart failure. 275n comparison to MSC-derived exosomes, exosomes derived from ESCs exhibited comparable therapeutic effect for cardiac conditions.In terms of protection of cardiomyocytes, Khan et al. discovered that the ESC-derived exosomal miR-294 could improve cardiomyocyte survival, promote neovascularization and inhibit fibrosis after MI, thereby augmenting post-MI cardiac function. 276n addition, Tavakoli Dargagni et al. demonstrated that ESCderived exosomes could alleviate doxorubicin-induced cardiotoxicity by inhibiting TLR4-NLRP3-mediated pyroptotic cell death in cardiomyocytes. 277Similarly, Singla's team showed that ESCderived exosomes could improve cardiac remodeling by enhancing anti-inflammatory M2 macrophages and reducing inflammation-induced pyroptosis. 278In terms of management of heart failure, Pang et al. exhibited that ESC-derived exosomes could attenuate heart failure, improve cardiac function and promote myocardial angiogenesis through the FGF2 signaling in a transverse aortic constriction-induced heart failure model. 279sing a coronary artery occlusion-induced heart failure model, Kervadec et al. showed that exosomes secreted by ESC-derived cardiovascular progenitors could recover cardiac functions such as reduced left ventricular end-systolic and end-diastolic volumes. 280inally, the same research group later demonstrated that exosomes derived from more readily available cell sources, e.g., iPSCs, were capable of cardioprotective effects similar to those offered by ESC-derived ones. 281ther important subtypes of SC-Exo, such as iPSC-exo, HSC-exo and EPC-exo, have also exhibited cardio-protective effects.For example, exosomes secreted by iPSCs could exert cytoprotective effects on maintaining intracellular Ca 2+ homeostasis and promoting cardiomyocyte survival, thereby improving recovery from MI. 282 HSC-exo could reduce the cardiac injury-related indices and the degree of cardiac fibrosis while elevating the ejection fraction in an animal model of heart failure. 283In addition, systemic infusion of HSC-derived exosomes could improve ischemic cardiomyopathy in a rat model of acute MI, with additional benefits in treating the side effects such as kidney damage. 284Modification of HSCs using sonic hedgehog (Shh), an angiogenic factor, could preserve cardiac function after acute MI by delivery of exosomal Shh to ischemic myocardium. 285Ke et al. proved that EPC-derived exosomes could enhance the proliferation and angiogenesis of cardiac fibroblasts by activating mesenchymal-endothelial transition and decreasing the expression of HMGB1 286 and later revealed that the exosomal miR-218-5p and miR-363-3p from EPC-derived exosomes could ameliorate MI by targeting the p53/JMY pathway. 287In an interesting study by Yue et al., IL-10 deficiency-induced systemic inflammation was found to compromise the reparative properties of EPC-derived exosomes on myocardial repair by upregulating integrin-linked kinase (ILK) enrichment in exosomes, and ILK-mediated activation of NF-κB pathway in recipient cells. 288n terms of treatment of thoracic disorders, Liu et al. proved that human ESCs-derived exosomes could alleviate inflammation, prevent excessive collagen deposition and preserve alveolar architecture in the lungs of mice with bleomycin-induced pulmonary fibrosis. 289This was achieved by the exosomal mi-17-5p targeting thrombospondin-2.Similarly, Zhou et al. demonstrated that the exosomal miR-302a-3p from iPSC-derived exosomes could suppress M2 macrophages via TET1, thereby mitigating pulmonary fibrosis. 290Liu et al. showed that EPCderived exosomes could inhibit pulmonary artery smooth muscle cells proliferation and their resistance to apoptosis by regulating the Mitofusin-2 and Ras-Raf-ERK1/2 pathways, thereby acting as a potential therapeutic candidate for the treatment of pulmonary arterial hypertension. 291Two independent teams both revealed that human EPC-derived exosomes could improve outcomes of the LPS-induced acute lung injury partially through the delivery of miR-126 into the injured alveolus. 292,293Zhang et al. found that EPC-derived exosomes could improve the bioactivity of pulmonary microvascular endothelial cells and protect them from hyperoxic injury in the developing lung vasculature, thereby contributing to the treatment of bronchopulmonary dysplasia. 294oreover, Montay-Gruel et al. demonstrated that human ESCderived exosomes could improve the adverse late normal tissue complications associated with exposure of the lungs to ionizing radiation, such as those encountered during postoperative treatment of lung cancer. 295OLOGY AND SC-EXO THERAPY Chronic kidney disease (CKD) is a syndrome characterized by persistent changes in kidney structure, function, or both, affecting 10−14% of the global population. 296The most common pathological feature and final manifestation of CKD is some form of renal fibrosis.Kidney fibrosis occurs when wound healing is deregulated, which leads to excessive accumulation of ECM proteins, such as collagen and fibronectin.In their study, Liu et al. discovered that bone marrow MSC-derived exosomes could alleviate vascular calcification, a detrimental indicator of morbidity and mortality in CKD. 297This was achieved through exosomal miR-381-3p by targeting NFAT5, which was further verified in severe arterial calcification in dialysis patients.In alternative studies, several groups demonstrated the capacity of bone marrow MSCderived exosomes in treating renal fibrosis, each with a distinct mechanistic interpretation.In a cellular study, Yin et al. found that exosomes could prevent TGF-β1-induced epithelial-mesenchymal transition of renal tubular epithelial cells by transporting Nedd4L, which activates autophagy.In a 5/6 subtotal nephrotomy rat model, Liu et al. revealed that exosomes could improve renal function and reduce fibrotic size by regulating the Smurf2/Smad7 axis. 298In a unilateral ureteral occlusion-induced interstitial fibrosis mouse model, Lu et al. demonstrated that exosomes could improve renal fibrosis by reducing the polarization of M1 and M2 macrophages by activating EP2 receptors. 299cute kidney injury (AKI) and CKD are closely connected, with each a risk factor for developing the other.Renal IRI is a leading cause of AKI and acute kidney failure. 300Lim et al. proved that exosomes from iPSC-derived MSCs could correct serum creatinine level, tubular necrosis, apoptosis, inflammatory cytokine production, and oxidative stress in AKI mice by activating the ERK1/ 2 signaling pathway. 301In a similar work by Zhang et al., the exosomal miR-21-5p from EPCs were found to alleviate sepsisinduced AKI by inhibiting RUNX1 expression in CLP rats. 302

OTORHINOLARYNGOLOGY AND HEAD & NECK SURGERY AND SC-EXO THERAPY
Hearing loss is the most common sensory deficit worldwide, affecting nearly 20% of the global population. 303The causes of sensorineural hearing loss (SNHL) can be very diverse, such as presbycusis, ototoxic medication-induced, noise-induced, and idiopathic sudden SNHL.Tsai's team demonstrated that umbilical cord MSC-derived exosomes could rescue the loss of outer hair cells and repair cochlear damage in cisplatin-induced hearing loss. 304The underlying mechanism for the cochleaprotective effects is mediated by the miRNAs (e.g., miR-125a-5p and miR-125b-5p) and remodeling factors (e.g., fibronectin and galectin-3).
Cochlear IRI is one of the main reasons for idiopathic sudden and noise-induced SNHL, which can lead to irreversible damage of sensory hair cells and bipolar cochlear spiral ganglion. 305Its pathophysiology includes oxidative stress, excess cell death and dysregulated inflammation.Hao et al. discovered that exosomes derived from miR-21-overexpressing NSCs could prevent hearing loss from IRI by inhibiting the inflammatory process in the mouse cochlea. 306This was evidenced by a reduced auditory brainstem response threshold, upregulated IL-10 and downregulated TNF-α and IL-1β.
Hypothyroidism is a very common disease which could result from thyroidectomy or radioactive ablation to treat hyperthyroidism or thyroid cancer. 307Stem cell therapy and stem cell-derived exosome therapy have emerged as a promising management for hypothyroidism through thyroid regeneration.Using an in vitro culture system of thyroid lobes, Degosserie et al. suggested that EPC-derived exosomes could facilitate thyrocyte organization into thyroid follicles and lumen expansion (i.e., folliculogenesis), which was promoted by laminin-α1. 308emporomandibular joint (TMJ) disorders are the second most common musculoskeletal condition affecting 31% of adults and 11% of children. 309Like other synovial joints in the body, TMJ is also prone to OA, sharing common pathophysiological processes (Section 3.2).Zhang and co-workers proved that MSC-derived exosomes could alleviate TMJ OA in an immunocompetent rabbit model by attenuating inflammation and restoring matrix homeostasis. 310The exosome-mediated joint repair was attributed to adenosine activation of Akt, ERK and AMPK signaling, as well as enhanced s-GAG synthesis.
OPHTHALMOLOGY AND SC-EXO THERAPY Acquired optic neuropathy is a major cause of blindness in adults, and has various etiologies, such as vascular, inflammatory, traumatic, toxic, compressive, and nutritional etiologies.Retinal ganglion cell (RGC) loss is the hallmark of optic neuropathies, via multiple cell death pathways. 311Mead and Tomarev found that bone marrow MSC-derived exosomes could promote survival of RGCs, and regeneration of their axons, in a rat optic nerve crush model. 312The exosomal neuroprotective and neuritogenic effects were accomplished through exosomal miRNAs, demonstrating a cell-free potential for traumatic and degenerative ocular diseases.
Retinal degenerative diseases (e.g., age-related macular degeneration and retinitis pigmentosa) are the leading cause of bilateral irreversible vision loss worldwide. 313It is characterized by progressive degeneration of photoreceptors, RGCs or retinal pigment epithelium (RPE) cells.Bian's team discovered that exosomes derived from NSCs could preserve photoreceptors, visual function and prevent thinning of the outer nuclear layer in an RCS retinal degeneration rat model. 314This was achieved by marked inactivation of microglial inflammation via exosomal targeting of TNF-α, IL-1β and COX-2.In two consecutive studies, Gao's team showed that ESC-derived exosomes could alleviate retinal degeneration by enhancing the proliferation and retrodifferentiation of retinal Müller cells as replacement retinal neuronal precursors.On one hand, this was accomplished by regulating the expression of Oct4 in Müller cells through exosomal HSP90. 315On the other hand, activation of the Wnt signaling pathway by delivering BDNF protein to Müller cells also played an important role. 316Furthermore, using a rat model of inherited retinal degeneration, Park et al. demonstrated that both subretinal and intravitreal injection of human HSC-derived exosomes could provide functional rescue of a degenerating retina. 317ailed healing of corneal defect often leads to corneal blindness which has been reported as second only to cataract in the leading causes of blindness. 318The most severe and recalcitrant cases would need corneal transplantation.Wang et al. compared exosomes derived from iPSCs and MSCs as therapeutic providers for the treatment of corneal epithelial defects. 319It was found that both types of exosomes could promote proliferation, cell cycle progression and migration while inhibiting apoptosis in vitro, and accelerate corneal epithelium defect healing in vivo.More importantly, the iPSC-derived exosomes had a stronger therapeutic effect than the MSC-derived exosomes.
Corneal transplantation is one of the most successful forms of solid organ transplantation.However, graft rejection can occur in up to 90% of high-risk recipients. 320Both innate and adaptive immunity are the predominant reason for graft failure.Immunosuppressive drugs have shown only partial effectiveness.Jia et al. showed that MSC-derived exosomes could cross biological barriers and prolong graft survival time in a rat model of corneal allograft rejection. 321This was likely caused by inhibition of the infiltration of CD4 + and CD25 + T cells and the reduction of IFN-γ and CXCL11 via the Th1 signaling pathway.

OBSTETRICS AND GYNECOLOGY AND SC-EXO THERAPY
Primary ovarian insufficiency (POI), or premature ovarian failure (POF), is defined as loss of ovarian function before the age of 40.Non-genetic causes of POI include autoimmune disorders, metabolic conditions, infections, and iatrogenic procedures (e.g., chemotherapy, radiotherapy, and surgery).Women with POI suffer from various complications, such as osteoporosis, infertility, cardiovascular disorders and depression. 322Although promptly initiating hormone replacement therapy is critical to control these symptoms and complications, it fails to restore ovarian function.Currently-tested experimental therapies include mitochondrial activation, in vitro activation, stem cell therapy, and exosome therapy. 323he work conducted by Li et al. showed that umbilical cord MSC-derived exosomes could improve ovarian function in a cyclophosphamide (CTX)-induced POI mouse model. 324The SCexo therapy not only restored ovarian function-related hormone levels and the number of ovarian follicles, but also improved the reproductive ability of POI mice.In addition, the exosomes promoted the proliferation of ovarian granulosa cells (GCs) by regulating the Hippo pathway, and the effect was neutralized by a YAP inhibitor.Similar results were obtained using exosomes from iPSC-derived MSCs. 325The work performed by Ding et al. illustrated that umbilical cord MSC-derived exosomes could restore ovarian phenotype and function in a POI mouse model, promote proliferation of CTX-damaged human GCs and oocytes, and alleviate ROS accumulation by delivering exosomal miR-17-5p and targeting its downstream mRNA SIRT7. 326It was further elucidated that miR-17-5p down-regulated PARP1, γH2AX, and XRCC6 expression by inhibiting SIRT7.Lastly, the work completed by Yang et al. revealed that bone marrow MSC-derived exosomes could recover the estrus cycle, increase the number of basal and sinus follicles, increase estradiol E2 and anti-Mullerian hormone levels, and reduce follicle stimulating hormone and luteinizing hormone levels in a chemotherapy-induced POF rat model. 327echanistically, this was achieved by exosomal miR-114-5p that targets PTEN.

FROM PRECLINICAL STUDIES TO CLINICAL TRIALS OF EXOSOME THERAPY
Many preclinical studies, as discussed in Sections 3 to 11, have confirmed the advantages of MSC-derived and NSC-derived exosomes to treat many diseases spanning the subspecialties of surgical practice.Without restricting the scope to stem cellderived exosomes only, many clinical trials have demonstrated the role of exosomes to be twofold: biological markers and therapeutic agents.A search on ClinicalTrials.govusing 'exosome therapy', 'exosome treatment', and 'exosome' as keywords generated 188 records.However, only 60 (32%) of these directly relate to interventional studies using exosomes as therapeutic agents (Table 5).The rest, especially oncology-related trials, mostly used exosomes as biomarkers, such as key players during disease pathogenesis (e.g., NCT04288141, NCT04154332), diagnostic markers and guidance before treatment (e.g., NCT04629079, NCT03791073, NCT05451342, NCT03432806), monitoring indices and predictive tools for treatment efficiency (e.g., NCT05427227, NCT04499794, NCT04852653, NCT05370105, NCT03800121, NCT05370105, NCT05328089), and prognostic indicators after treatment (e.g., NCT06026735, NCT05705583, NCT05411445, NCT04167722, NCT05575622).The clinical applications of exosomes as biomarkers have been extensively explored in other reviews [328][329][330][331][332] and are therefore beyond discussion in this review.In terms of the clinical characteristics of the 60 clinical trials on exosome therapy (Table 5), there are several highlights worth mentioning.
Firstly, the spectrum of diseases covered is very broad, as both surgical and medical conditions are included.These include many surgical disorders discussed in Sections 3 to 11, such as orthopedic diseases (osteoarthritis of the knee in NCT05060107, bone loss in NCT04998058, and intervertebral disc degeneration in NCT04849429), neurosurgical diseases (ischemic stroke in NCT03384433 and Alzheimer's disease in NCT04388982), plastic surgical diseases (cutaneous wound healing in NCT02565264 and NCT05475418), general surgical diseases (liver cirrhosis in NCT05871463), cardiothoracic diseases (myocardial infarction in NCT05669144), and ophthalmology diseases (retinitis pigmentosa in NCT05413148).In other words, some preclinical studies have not yet developed into clinical trials.These include, but are not limited to, exosome therapy for fracture 333 spinal cord injury 334 traumatic brain injury 335 acute liver injury 336 and hearing loss 304,306 which might serve as future directions for exosome therapy-related clinical trials.
Secondly, an increasing number of clinical trials focused on two medical conditions, i.e., COVID-19 (16 trials, 27%) and cancer (5 trials, 8%).MSC-derived exosomes can manage viral infection and lung damage in COVID-19 through both reparative actions and regenerative effects. 337The former manifests as blockage of viral entry and replication, and suppression of the cytokine storm, whereas the latter as prevention of inflammation, and fluidclearance and restoration of lung permeability.In addition, exosomes can be engineered into a drug (e.g., CD24, a potent immune regulator) delivery platform 338 and even a vaccine 339 to combat COVID-19.In contrast to the MSC-derived exosomes for COVID-19 management, exosomes used for cancer treatment mainly rely on non-stem cells and cargo engineering.This is partially because MSC-derived exosomes demonstrate controversial effects on tumorigenesis and metastasis. 340,341][344][345][346] Finally, among the 40 clinical trials using stem cell-derived exosomes for disease treatment, 38 (95%) used MSC and 2 (5%) used iPSC as the cellular source for exosomes.However, this differs significantly from the preclinical studies discussed in Sections 3 to 11.The lack of use of NSC-derived exosomes in clinical trials might be partially because of the supply constraints of their parental cells. 347Currently, NSCs can be obtained from three sources 348 (Fig. 1a): 1, isolation from primary CNS tissues (e.g., adult and fetal brain); 2, differentiation from pluripotent stem cells (e.g., iPSCs and ESCs); 3, reprogramming of somatic cells (e.g., fibroblasts and blood cells) to iNSCs. 349Recent studies have developed fibroblastderived iNSCs, opening a new window for obtaining exosomes from NSC-like cells.These iNSC-exo could not only promote cell survival and proliferation no less than NSC-exo in vitro 350,351 but also enhance recovery after ischemic stroke 148 and mitigate ADlike phenotypes in preclinical models. 190Therefore, iNSCs might be an excellent cellular source to produce clinical-grade exosomes in clinical trials.
In summary, the progression from preclinical studies to clinical trials of exosome therapy has been expeditious.However, issues like insufficient clinical indications for exosome treatment and limited sources for parental stem cells remain to be addressed.In addition, once the limitations (Table 1) in upscaling of manufacturing, compliance with good manufacturing practice, and regulatory framework are overcome 329 stem cell-derived exosome therapy will soon be incorporated into clinical practice and serve at the patient's bedside.

CONCLUSION AND FUTURE PERSPECTIVES
Exosomes have been pursued recently as a cell-free alternative to stem cell-based therapy.ESC-, iPSC-, HSC-, MSC-, NSC-and EPCderived exosomes are of particular interest, partially due to the pluripotency or multipotency of their parental cells.After going through production and purification with or without modification, stem cell-derived exosomes have demonstrated tremendous potential in treating numerous diseases encountered during surgical practice.These are exemplified by disorders in orthopedic surgery (e.g., fracture, osteoarthritis, and spinal cord injury); neurosurgery (e.g., ischemic stroke, traumatic brain injury, and Alzheimer's disease); plastic surgery (e.g., wound healing); general surgery (e.g., acute liver injury); cardiothoracic surgery (e.g., myocardial infarction); urology (e.g., chronic kidney disease); head and neck surgery (e.g., sensorineural hearing loss); ophthalmology (e.g., acquired optic neuropathies), and gynecology (e.g., primary ovarian insufficiency).Mechanistically, the diverse therapeutic effects of stem cell-derived exosomes are achieved through disease-specific cellular and tissue responses (e.g., tissue regeneration, anti-inflammation, anti-cell death, immunomodulation, and anti-oxidative stress) and tissue-specific molecular signaling pathways (e.g., Wnt/β-catenin, PTEN/PI3K/Akt/HIF-1α, MAPK, and JAK/STAT pathways).Collectively, stem cell-derived exosome therapy has been proven to be a potent and versatile surrogate to stem cell therapy in the surgical arena.Future emphasis of clinical applications of stem cell-derived exosomes should be placed on various nodes of this therapeutic pipeline.Firstly, targeting the pretherapeutic large-scale production of exosomes, a high-throughput cellular source as well as a reproducible and scalable production and isolation protocol are required.Compared to the static system growing monolayer cells, dynamic system in the form of bioreactor, e.g., hollow-fiber bioreactor and stirred tank bioreactor, can increase the efficiency by producing copious cells and exosomes in a short period of time.However, the phenotype of parental cells and derived exosomes might change due to physical and shear stress encountered in a reactor.Thus, the working parameters of the bioreactor must be optimized to facilitate large-scale production of stem cell-derived exosomes.Secondly, targeting the therapeutic modality of exosomes, delivery methods other than systemic administration need to be explored.When delivered through the venous system, exosomes are rapidly cleared from blood circulation and accumulate in the liver, spleen and lungs, which can be overcome by local delivery.Various biomaterials have been recently used to protect, assist and augment locally delivered exosomes to maximize their therapeutic effects.These biomaterials could be designed according to their sources (e.g., natural, synthetic, and hybrid polymers), format (e.g., scaffold, patch, spray, and microneedle), and responsiveness (e.g., temperature, pH, and protein), thereby allowing disease-specific customization.Lastly, targeting the therapeutic indications of exosome therapy, more diseases than the ones discussed in this review should be included into future preclinical studies and clinical trials.For example, airway inflammatory conditions (e.g., allergic rhinitis and asthma) could be suitable candidates for exosome therapy, considering the immunomodulatory effect of MSC-derived exosomes.Disorders that are best treated using surgical implants (e.g., cochlear implant, intraocular lenses, and contraceptive intrauterine devices) could be managed in the form of implantbased local release of exosomes.In addition to primary diseases, secondary conditions including surgical operation-and general anesthesia-related complications (e.g., cognitive impairment, wound paresthesia, and malignant hyperthermia) might become therapeutic targets of exosome therapy.Collectively, efforts to upscale exosome production in conjunction with multimodal exosome delivery will accelerate the clinical applications of stem cell-derived exosomes in a rapidly expanding disease spectrum.

Fig. 1
Fig. 1 Illustration of the upstream measures of exosome therapy (figure generated using Autodesk 3ds Max 2023).a production and purification of exosomes (MSCs and NSCs are used as examples for multipotent stem cells).b content of natural exosomes.c modification of exosomes.(BM bone marrow, DC dendritic cell, IAC immunoaffinity chromatography, iPSC induced pluripotent stem cell, MHC major histocompatibility complex, miRNA microRNA, MSC mesenchymal stem cell, MVB multivesicular body, NSC neural stem cell, SEC size-exclusion chromatography, UC umbilical cord)

Fig. 3
Fig. 3 Mechanisms of stem cell-derived exosome therapy (figure generated using Adobe Photoshop 2023 and Adobe Illustrator 2023).a activation and regulation of various signaling pathways.b disease-specific cellular and tissue responses

Table 2 .
Stem cell-derived exosomes for the treatment of diseases in orthopedic surgery and related specialties

Table 3 .
Stem cell-derived exosomes for the treatment of diseases in neurosurgery and related specialties

Table 3 .
continued work conducted by Xing et al. focused on atherosclerosis.The adipose MSC-derived exosomal miR-342-5p was shown to protect endothelial cells against atherosclerosis by targeting PPP1R12B in a H 2 O 2 -challenged HUVEC model. 268The work conducted by Peng et al. and Gao et al. focused on MI.The exosomal miR-25-3p from MSCs could alleviate MI by targeting pro-apoptotic proteins and EZH2 in an OGD cardiomyocytes model and left anterior descending artery ligation animal model.

Table 5 .
Clinical trials of exosome therapy

Table 5 .
continued Data obtained from ClinicalTrials.gov using 'exosome therapy' , 'exosome treatment' , and 'exosome' as keywords, as of 2023-09-08.The categorization of diseases follows the system by Clinical Trials.govAD Alzheimer's disease, ARDS acute respiratory distress syndrome, CM conditioned medium, COPD chronic obstructive pulmonary disease, DC dendritic cell, DM diabetes mellitus, ELBW extremely low birth weight, HCC hepatocellular carcinoma, IBS irritable bowel syndrome, iPSC induced pluripotent stem cell, LDLR low-density lipoprotein receptor, MI myocardial infarction, MPC mesenchymal progenitor cell, MSC mesenchymal stem cell, NSCLC non-small cell lung cancer, OA osteoarthritis, PCOS polycystic ovary syndrome, PRP platelet-rich plasma, siRNA small interfering RNA Clinical applications of stem cell-derived exosomes Tan et al.